I recd. so many requests from people who did not get my earlier post that I
decided to save time by re-posting it. At the outset I would like to state
that I am only a satisfied user of the above vectors and am not associated with the company that markets them.
pow
Hi netters, I would like to report the availability of a set of reporter gene vectors that have some distinct advantages over those currently in use for the purpose of analysis of eukaryotic promoters & enhancers. The vectors , SV40-Syncat, Syncat I, Syncat II, SV40-pFlash, pFlash I and pFlash II, are as their names suggest CAT & Luciferase (Luc) reporter gene vectors. What distinguishes them from other vectors in this category is that these vectors produce near-zero background. This feature is due
to the fact, that they use a modified SV40-t-intron as the donor for the poly-adenylation signal. The wild type SV40-t-intron is the generic donor for this function and is widely used in a variety of reporter gene vectors, eg. pCAT- basic, pCAT-promoter, pGL-basic and pGL-promoter (Promega). However one little known fact about the wild-type-t-intron is that it contains cryptic enhancer sequences that produce significant background activity, thereby raising the threshold of sensitivity. Until now this
feature was not a serious limitation since scientist were analysing strong promoters/enhancers that produced high signal to noise ratios. However the focus in the past couple of years has shifted to the analysis of weak promoters, or promoters that have very low basal transcription rates but are specifically induced to high levels by cytokines, or promoters that function only in cells that are very poorly transfectable. Analysis of such regulatory elements requires systems of high sensitivity as wel
l as specificity. The Syncat & pFlash series of vectors provide precisely these capabilities, since they contain a t-intron polyA signal that has been modified to be devoid of cryptic enhancer activity. The 2nd. feature of these vectors is the choice of the heterologous promoter in Syncat II and pFlash II. These vectors use the well characterized HSV-tk TATA box containing minimal promoter situated immediately upstream of the reporter gene. The HSV-tk is a widely used basal promoter that has been do
cumented to produce no spurious interactions with enhancers of interest. By contrast vectors like pCAT-promoter or pGL-promoter use the SV40 promoter. The SV40 promoter contains an atypical TATA-box and more seriously has been documented to spuriously repress certain cytokine inducible enhancers (Benech, et.al.. J.Exp.Med. 1992, Vol. 176: 1115-1123). I should know, because I am one of the authors of that paper and this defect in the pCAT-promoter vector cost me 6 months of time and nearly resulted i
n our being scooped by a competing lab. Other features of these vectors include a very versatile multiple cloning site upstream and downstream of the reporter gene cassette, flanked by T3 & T7 promoter sequences for direct sequencing and creation of unidirectionally deleted mutant libraries as well as f1 origin of replication for ssDNA recovery and site-directed mutagenesis. The combination of these features enabled me to perform all my DNA manipulations from cloning of a 5 kb genomic 5U flanking r
egion upstream of the reporter gene ---to--- creation of nested deletion mutant libraries followed by sequence verification ---to--- ssDNA-site-directed mutagenesis and transfection of each construct --- ALL IN THE SAME REPORTER GENE VECTOR I STARTED WITH. The time savings alone were upto 50%. These vectors are available from SynapSys Corp. To get more info about them send E-mail to :- #
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synapsys at world.std.com. #